The oral microbiome has been gaining a lot of attention as a key player in nitric oxide (NO) metabolism, and accordingly in cardiometabolic health. Commensal oral bacteria reduce exogenous (dietary) and endogenous nitrate to nitrite, which is converted to NO, a signaling molecule that regulates vascular tone, inflammation and insulin sensitivity. Randomized clinical trials have shown that eliminating the oral microbial flora through the use of antiseptic mouthwash decreases systemic NO bioavailability with concomitant loss of insulin sensitivity and increased blood pressure. Nitrite can also be further reduced by oral bacteria into inorganic nitrogen compounds, such as ammonia via Dissimilatory Nitrate Reduction to Ammonia (DNRA) pathway, which can play important biological roles in oral health; these pathways could also impact systemic NO bioavailability. Our recent publication from the San Juan Overweight Adults Longitudinal Study (SOALS) shows that regular use of over- the-counter mouthwash is significantly associated with increased risk of pre-diabetes/diabetes over a three-year follow-up, independently of major diabetes risk factors. The oral microbiome has not yet been characterized in direct relation to its nitrate reducing capacity and its role in NO bioavailability and related oral and cardiometabolic health outcomes. Accordingly, we propose to evaluate the following specific aims. (1) To identify oral microbial profiles associated with nitric oxide bioavailability (serum nitrite levels). (2) To evaluate the longitudinal association of oral microbial profiles related to nitric oxide bioavailability with the development of hypertension, pre-diabetes/diabetes, dental caries and progression of periodontitis. (3) To evaluate the impact of mouthwash use on these microbial profiles and systemic NO bioavailability. In addition, we will also evaluate the association of oral microbial profiles with other components of oral nitrate metabolism (salivary nitrate, nitrite, nitrate reductase and DNRA activity). Modifiable lifestyle factors such as diet that may impact oral nitrate reducing bacteria and pathways will be assessed. Over 1,000 SOALS participants have pertinent high quality data and specimens available at baseline and 3-year follow-up visits (with 79% retention) to enable a timely cost-efficient study. Saliva and blood samples, oral and cardiometabolic clinical measures, and key covariates including detailed dietary, physical activity and oral hygiene measures have been collected. We will perform analysis of the salivary microbiome using 16S rRNA sequencing, and we will measure key components of endogenous and exogenous NO metabolism (nitrite and nitrate in serum and in saliva, nitrate reductase and DNRA activities in saliva). The results from this study will help define the interrelationships between oral bacteria, NO metabolism, cardiometabolic and oral health, and help understand pathways relating mouthwash use and cardiometabolic conditions. This study has a high potential public health impact, as the results will likely inform development of novel interventions for the modulation of the oral microbiome, which could be important for the prevention and control of oral and cardiometabolic diseases.